Evidence for a metameric pattern in the development of the chick humerus

Transcription

1 /. Embryol. exp. Morph. Vol. 61, pp , 1981 Printed in Great Britain Company of Biologists Limited 1981 Evidence for a metameric pattern in the development of the chick humerus By TRENT D. STEPHENS AND TERESA R. McNULTY Central Laboratory for Human Embryology, Departments of Pediatrics and Biological Structure, University of Washington SUMMARY It has been proposed that the wing bud is induced by some axial influence at a specific confined location and that the ZPA is the residual influence of such induction. The purpose of the present investigation was to test this hypothesis. Tantalum foil barriers were placed lateral to the mesonephric duct and parallel to the long axis of the embryo in the wing field of stage12 to 15 chick embryos. These barriers blocked the somatopleure's communication with more medial tissues at specific somitic levels. The results of these experiments demonstrate that (1) the limb is not induced at one specific point, (2) portions of the humerus appear to be induced segmentally along the entire limb field and (3) the ZPA is not induced by axial structures. We propose a model of wing development suggesting that the humerus is induced as several separate components which then fuse to form the definitive bone. INTRODUCTION The chick wing bud develops adjacent to somites 152 beginning at stage 16 (Hamburger & Hamilton, 1951) and there is considerable evidence that the initial events in wing development are in some way influenced by adjacent axial structures (Kieny, 19; Kieny, Mauger & Sengel, 192). This inductive influence of the axial tissues may be blocked by the imposition of a physical barrier (Hamburger, 1953) between the somites and lateral plate (Murillo Ferrol, 1965; Sweeney & Watterson, 1969). It has been thought that the inductor may in some way relate to the establishment of the zone of polarizing activity (ZPA) (Balcuns, Gasseling & Saunders, 19). The ZPA wasfirstdescribed by Saunders and Gasseling (1968) as an area of mesoderm in the proximal, postaxial portion of the developing wing bud which, when grafted to the preaxial border of a wing, induced mirror image duplications of the wing tip. Summerbell (199) has proposed that the ZPA may be a selfpropagating gradient laid down during early inductive events. Fallon & Crosby (19) said of the ZPA, 'possibly only a few cells may determine not 1 Author's address: Department of Pediatrics, RD2, University of Washington, Seattle, WA 98195, U.S.A. 2

2 192 T. D. STEPHENS AND T. R. McNULTY only that a limb bud will grow out at a particular level on the body axis, but also where the posterior border of that limb will be'. The suggestion made by Fallon & Crosby (19) that the wing may be induced by an influence confined to a site which occupies only a small portion of the entire limb field is given support by the work of Sweeney & Watterson (1969). They observed, 'complete absence of the right wing... in 1 of 11 specimens in which the anterior edge [of a tantalum foil barrier]... was inserted at the level of somite 18'. Sweeney & Watterson (1969) thus blocked only the postaxial half of the wing field but completely inhibited wing development. Further evidence in support of Fallon and Crosby's (19) suggestion was obtained by MurilloFerrol (1965) and Summerbell (199). They demonstrated that the preaxial half of the wing will not develop when blocked from interaction with the postaxial half. The remainder of MurilloFerrol's data (1965), however, does not agree with the idea of a small, postaxial location of the inductive influence. Short barriers placed in the middle of the limb field resulted in the induction of two limb buds, one posterior and one anterior to the barrier. This observation suggests that the limb may be induced from a region that is much broader than that proposed by Fallon & Crosby (19). The purpose of the present study was to repeat and expand upon the observations of Sweeney & Watterson (1969) and to account for the apparent discrepancies between their data and that of MurilloFerrol (1965). By doing this we hoped to explore the extent of inductive influence which establishes the limb field. The results of these experiments suggest that (1) the limb is not induced at one specific point, (2) anteriorposterior portions of the wing appear to be induced segmentally along the entire limb field and (3) the ZPA is not induced by axial structures. Based upon these points, we have developed a model suggesting segmental development of the humerus. MATERIALS AND METHODS White Leghorn chicken eggs (College Biological Supply Co., Bothwell, WA), which had been incubated for 6 h at 338 C (stages 1215; Hamburger & Hamilton, 1951), were prepared for surgery by removing 152 ml of albumin and cutting a hole in the side of the egg to allow access to the embryo. A few drops of sterile saline were placed over the embryo and the vitelline membrane was removed. The embryos were unstained and the experiments were conducted with the aid of a Wild M8 Stereomicroscope. The use of this microscope enabled adequate visualization of the unstained embryos. The total somites were counted with the first full somite posterior to the otic placode counted as somite No. 1. A longitudinal slit, four to eight somites long, was made through the somatopleure at specific somite levels just lateral to the right mesonephric duct with a finely sharpened tungsten needle. Any embryos

3 Evidence for a metameric pattern in the chick humerus 193 with severe bleeding were discarded. In 35 control specimens, the eggs were then sealed with cellophane tape and returned to the incubator. In 248 experimental embryos, a piece of tantalum foil (38 mm x 115 mm and bent at a 9 angle lengthwise to give a functional blocking area 154 mm x 115 mm) was inserted into the slit, thus blocking an average of six somites (range: four to eight). Only somites adjacent to the limb field were referred to in the results. Even though the remainder of the barrier blocked more caudal or cranial somites outside of the wing field, these did not appear to contribute to the observed abnormality. The eggs were sealed with cellophane tape and returned to the incubator. In 22 additional experimental embryos, foil barriers were inserted into the somatopleure perpendicular to the long axis of the embryo. The embryos were inspected 24 h later and the position of the foil was noted along with the presence or absence and size of the right wing bud. The eggs were again sealed with tape and returned to the incubator for an additional 6 days. The chicks were removed from the egg and fixed in 1 % phosphatebuffered formalin after a total incubation time of 9 or 1^ days. The overall morphology of the operated wing and foil location was recorded and representative specimens were photographed. The length and width of the stylopod, zygopod and autopod of both the operated wing and the contralateral unoperated control wing of the experimental and control embryos were measured and recorded. A drawing was then made of each operated wing with the position of the foil indicated. The chicks were decapitated, eviscerated and double stained with toluidine blue and alizarin red S according to the technique of Burdi and Flecker (1968), cleared with 1 % KOH and stored in a 2:2:1 mixture of glycerol, ethanol and benzyl alcohol. Glacial acetic acid (three drops/2 ml) was added to the 5 % ethanol destaining solution to facilitate the destaining. The stained chicks were scored for the presence or absence, size and polarity of each bony component of the wing. Each humerus, both experimental and contralateral controls, were measured for length, greatest proximal diameter, greatest distal diameter, and central diameter. The direction of tapering, proximal or distal, was also noted. RESULTS There were 24 of 35 control embryos and 162 of 248 experimental embryos that survived to 9 1 days. One out of the 24 controls had a reduced limb (the rest were normal) and 31 out of the 162 experimental embryos showed no wing malformations. There were 131 experimental embryos exhibiting some degree of limb reduction and 3 of these exhibited no wing on the operated side. Representative experimental chicks are shown in Fig. 1. Figures 2 and 3 illustrate some of the general trends in reduction defects following foil implants. The photographs exhibit stained and cleared experi

4 194 T. D. STEPHENS AND T. R. McNULTY Fig. 1. Nine and tendayold chick embryos following foil barrier implants at day 2\. Barriers blocking wing somites: (A) 151; (B) 1518; (C) 152; (D) 192; (E) 12; (F) 162. mental wings with various degrees of reduction. The drawings show the proposed pattern of loss for each photograph as well as the barrier location resulting in the deficient limb. The pattern of loss in each drawing was based upon the calculation of proximal, central and distal diameter and overall length as a percentage of the contralateral control. The pattern of loss in the remaining wing segments was based upon estimates of the deficient tissue. Figure 4 illustrates the pattern of bone loss in each reduced humerus. Tables 1 and 2 give the specific data for the figure. Table 1 gives the average dimensions of the humeri, expressed as a percentage of the contralateral controls and Table 2 gives the number of reduced humeri in each pattern group (AF in Figure 4 and the two tables) resulting from barrier implants at specific somite levels. The humeri could be divided into six distinct patterns of loss in addition

5 Evidence for a metameric pattern in the chick humerus 195 \! /.. '. Fig. 2. Patterns of wing skeletal reduction (dotted lines) following specific foil implants (cross hatching). Left diagrams indicate site of foil implantation. Center diagrams schematically illustrate the proposed pattern of skeletal reduction based upon percent of the contralateral control. Photographs at right are examples of limb reductions following foil implantation at the indicated levels. Foil implant blocking wing somites: (A) 1516; (B) 1518; (C) to a 'normal' group and a group with complete absence of the experimental humerus. Figure 4A consists of a group of seven humeri all of which exhibited a decrease in only the central diameter. These humeral reductions resulted from implants primarily adjacent to wing somites 1516 (Table 2). Figure 4B consists of the diagrams of 1 humeri exhibiting a decrease in both the central and proximal regions without decrease in length. These reductions resulted from implants primarily adjacent to wing somites 151 and 1518 (Table 2). There is a dramatic change from Fig. 4B to Fig. 4C. The latter consists of a group of 1 humeri all exhibiting a marked reduction of proximal tissue and considerably reduced in length as compared to the previous groups. These reductions resulted from implants adjacent to wing somites 1518 and 1519 (Table 2).

6 196 T. D. STEPHENS AND T. R. McNULTY if i Fig. 3. Patterns of wing skeletal reduction (dotted lines) following specific foil implants (cross hatching). Left diagrams indicate site of foil implantation. Center diagrams schematically illustrate the proposed pattern of skeletal reduction based upon percent of the contralateral control. Photographs at right are examples of limb reductions following foil implantations at indicated levels. Foil implant blocking wing somites: (A) 192; (B) 12; (C) 162. Figures 4D, E and F exhibit a pattern of loss similar to Figs 4A, B and C, but approximately reversed in reduction pattern as well as location of implant. Figure 4D, a group of 1 humeri, exhibits a decrease in the diameter of the central and distal regions of the bone. These reductions resulted from implants primarily adjacent to wing somites 12. Figure 4E is a rather small group, six of which resulted from postaxial blocks, and two resulted from preaxial blocks. The group exhibited a loss of proximal, central and distal diameter without a loss in length. There is a dramatic change from Fig. 4E to Fig. 4F, similar but opposite to that seen between Figs 4B and 4C. Figure 4F, a group of 14 humeri, resulted primarily from implants adjacent to wing somites 162 and 12. In addition to the 15 humeri illustrated in Fig. 4 and listed in Tables 1 and 2,

7 Evidence for a metameric pattern in the chick humerus 19 Fig. 4. Representations of deficient humeri falling into six basic patterns of loss. Each humerus was measured for length, proximal diameter, distal diameter and central diameter. In order to standardize the results, each dimension was expressed as a percent of the contralateral control humerus and then drawn in relation to a standard control outline. See Table 1 for specific data for each dimension. The proximal end of each humerus is indicated by a fragment of the scapula attached to the humerus. Table 1. Effects of tantalum foil barrier implants on the humerus* Pattern of lossf A B C D E F Complete absence of wing Normal wing No. of Humeri Length 94±2 92±1 5O±5 93 ±2 92±2 41 ±3 98±1 Proximal diameter 95±4 62±3 9±3 62±3 58± 96±2 Central diameter 64±4 63 ±5 2±9 9±3 62±6 1±6 ±2 Distal diameter 9±4 94±2 86±6 54±5 68 ±5 99±2 ± Standard error. * Average dimensions of humeri expressed as a percentage of the contraleral controls. t Patterns of loss (AF) taken from Fig. 4.

8 198 T. D. STEPHENS AND T. R. McNULTY Table 2. Effects of tantalum foil barrier implants on the humerus Position of foil barrier opposite wing somitest Pattern of Total loss* humeri A B C D E F Complete absence of humerus Normal humerus Totals * Pattern of loss: AF taken from Fig. 4. t Only the somites adjacent to the wing field are listed although the barrier usually extended cranially or caudally for a greater distance there were another 12 reduced humeri which were not categorized. One humerus was broken and could not be accurately measured. In one case the humerus developed inside the thoracic cavity and was excluded from the remaining data. One chick exhibited multiple leg and vertebral column malformations perhaps as a result of membrane interference and the wing was thus excluded from analysis. One other humerus was markedly shortened but did not exhibit any taper and thus could not be categorized. In addition to these isolated cases, there were two groups of humeri which could not be categorized. The first group, with three members (two with 12 blocks and one with a 1518 block), resulted in humeri that were reduced in every dimension, length and all three diameters. Furthermore, all three wings in this group were fused at the elbow. This latter phenomenon was not seen in any of the other 128 reduced limbs. The second group, with five members, consisted of humeri with abnormal extra tissue. Two exhibited proximal spikes, two proximal broadening and one distal broadening. In addition one humerus with proximal broadening exhibited a central spike and the other an ectopic bone lying parallel to the humerus. In the case with the distal broadening, the foil was embedded in the broadened cartilage. In two of the other four cases, the foil was noted embedded in the limb bud at the 24 h check, and in one other case the foil was caught in the membranes and had been moved. In addition to the above mentioned noncategorized humeri, there were wings with either normal humeri or absent humeri that should be mentioned. Of the 38 wings with normal humeri, seven were missing distal structures while 31

9 Evidence for a metameric pattern in the chick humerus 199 Table 3. Effects of tantalum foil barrier implants on the radius, ulna and digits* Wing somites blockedf Number of implants Radius Reduced Absent Total affected Ulna Reduced Absent Total affected Digit II Reduced Absent Total affected Digit III Reduced Absent Total affected Digit IV Reduced Absent Total affected * Each number is represented as a percent of the total implants at that level. t Only the somites adjacent to the wing field are listed although the: barrier usually extended cranially or caudal ly for a. greater distance were normal wings. Two preaxial blocks (151 and 1518) resulted in loss of the radius and reduction of the digits while retaining a normal humerus. Five postaxial blocks (12, 182(2), 192, 2) resulted in loss of the ulna and usually the fourth digit in the presence of a normal humerus. On the other hand, of the 4 wings with no humerus, three exhibited growth of distal tissue, a wing tip consisting of part of the ulna or radius and one or two reduced digits. All three of these latter wings resulted from barriers adjacent to wing somites Table 3 demonstrates the effect of the position of foil implants on the other components of the wing. The radius appeared to be most strongly affected by barriers positioned adjacent to somites 1518, 1519, 152 and 162. The ulna appeared to be most strongly affected by barriers positioned adjacent to somites 152, 162 and 12. Digit II appeared to be strongly affected by barriers positioned adjacent to somites 1519, 152, 162; digit III appeared to be affected primarily by barriers positioned at levels 152 and 162. Digit IV appeared to be most strongly affected by barriers positioned opposite wing somites 152, 162 and

10 2 T. D. STEPHENS AND T. R. McNULTY B Fig. 5. (A) Tendayold chick embryo with two right wings resulting from a foil barrier implanted perpendicular to the long axis of the embryo adjacent to somite 18. (B) Right wings of the chick embryo stained with alizarin red and toluidine blue shown in (A). The preaxial reduced wing (a) exhibits only a proximal portion of the humerus. The postaxial reduced wing exhibits a distal portion of the humerus (b), the ulna and digits III and IV. Figure 5 illustrates the result of a foil barrier implanted perpendicular to the long axis of the embryo. Figure 5B depicts the stained specimen with the humerus divided into a proximal, preaxial portion and a distal, postaxial portion. Fifteen of 22 embryos with the barrier placed perpendicular to the long axis of the embryo survived the surgery. Eleven embryos exhibited split humeri (Fig. 5) and the remaining four exhibited multiple tissue nodules. The limb polarity was considered abnormal if any structure which developed

11 Evidence for a metameric pattern in the chick humerus 21 distal to the elbow was abnormal in its positioning or orientation. Of 94 malformed wings exhibiting growth beyond the elbow, 11 were found to have some degree of abnormal polarity. Only one of the 'abnormal' limbs exhibited what might be considered mispositioning of the digits consisting of what appeared to be two numberiv digits on the same wing. The remaining ten consisted of wings in which the elbow was bent in an abnormal direction (cf. Fig. 3B). Six of the 11 resulted from postaxial blocks (i.e. somites 162, 12 or 182) and five resulted from preaxial blocks (i.e. somites 1519 or 1518). DISCUSSION One purpose of the present study was to repeat and expand upon the observations of Sweeney & Watterson (1969) and to account for the apparent discrepancies between their data and that of MurilloFerrol (1965). Our results suggest that the limb is induced at several positions along its anteriorposterior length and not at one small postaxial location. This data contradicts the data of Sweeney & Watterson (1969) which led to the suggestion of a postaxial induction of the entire wing, but supports the observations of MurilloFerrol (1965) suggesting a broader influence. Another purpose of the present study was to test the hypothesis that the ZPA is a postaxial residue of the initial induction of the limb. Our data suggest that the ZPA is the source of an influence which is independent of any axial induction of the limb. We present here two pieces of evidence that the ZPA is not induced by axial structures, at least after stage 12 of development. First, of 94 wings exhibiting growth beyond the elbow, only 11 were found to have some degree of abnormal polarity. Of the 11, 6 resulted from postaxial blocks and 5 resulted from preaxial blocks. Therefore, barriers separating the postaxial limb field (ZPA) from the somites had no more effect on polarity than barriers separating the preaxial (nonzpa) region of the limb field from the somites. Second, barriers blocking somites 182 resulted in limbs with a portion of the humerus radius and often digits II and III present (Fig. 6 A). On the other hand, barriers placed perpendicular to the long axis of embryos between somites 1 and 18 resulted in two partial limbs (Fig. 6B). The partial limb postaxial to the barrier developed a partial humerus, an ulna and digits III and IV. The partial limb preaxial to the barrier developed only a portion of the humerus. Therefore, a block parallel to the long axis of the embryo appears to allow more distal pattern formation in preaxial tissue than a block perpendicular to the long axis at the same level. This would suggest that with a parallel barrier (Fig. 6 A), a postaxial influence (ZPA) is not inhibited and can facilitate development of preaxial structures. On the other hand, a perpendicular barrier (Fig. 6B) blocks the postaxial influence from reaching the preaxial tissue and thus prevents the ZPA from facilitating the subsequent distal development of preaxial structures. Three pieces of data suggest that the proximal and distal humeral segments

12 22 T. D. STEPHENS AND T. R. McNULTY ZPA ZPA Fig. 6. Schematic representation of the influence of the ZPA upon wing pattern formation. (A) Illustrates the effect of placing a barrier between the presumptive ZPA and the adjacent wing somites (182). (B) Illustrates the effect of placing a barrier between the presumptive ZPA and the preaxial limb tissue perpendicular to the long axis of the chick between somites 1 and 18. described above exhibit a preaxial and postaxial tapering, respectively (cf. Figs 2, 3 and ). First, it may be noted that the two humeral segments resulting from a perpendicular foil implant consisted of entirely different regions of the bone (Fig. 5B). The humeral segment which developed preaxial to the barrier was the proximal head of the bone with a gradual distal tapering. The partial humerus which developed postaxial to the perpendicular barrier consisted of the distal end of the bone and a gradual proximal tapering. Second, the trend

13 Evidence for a metameric pattern in the chick humerus 23 Fig.. (A) The proposed pattern of humeral components based upon observations of foil barrier induced limb deficiencies. The double hatched area was obtained as a mean from loss pattern Fig. 4 A (showing tissue lost) and assumed to be preaxial because of the preaxial locations of the foil producing the group. The white area was obtained as a mean from loss pattern Fig. 4F and the stippled, area from Fig. 4C (both showing tissue present). The tapering direction, postaxial or preaxial, in the white and stippled areas was assumed based upon postaxial or preaxial location of implants and also upon Figure 5B (see discussion). (B) Schematic representation of 'sclerotomal' patterns of referred pain from a human humerus (After Inman & Saunders, 1944). of reduction in Fig. 4 was proximal loss associated with preaxial barriers (Fig. 4 A, B and C) and distal loss associated with postaxial barriers (Fig. 4D, E, F). Third, the radius was more often missing in association with loss patterns A, B and C, while the ulna was seen to be lost with loss patterns D, E and F. These observations of humeral reduction trends have led us to postulate a model of humeral development. According to this model, the humerus develops, not as a single bone, but as a mosaic of several components which in some way unite to form the complete bone (Fig. A). These components are in turn induced, or at least influenced, by axial or paraaxial structures in a segmental fashion. The model does not propose that this influence emanates specifically from the somites (indeed, Chevallier, 198 has demonstrated that removal of somite mesoderm does not interfere with normal wing skeletal patterning), but rather that the foil barrier blocks the lateral migration of an influence of unknown identity. Hence, the model proposes the existence of some influence that stimulates the development of individual humeral segments which together form a complete bone. The fact that a barrier placed perpendicular to the embryo axis results in the

14 24 T. D. STEPHENS AND T. R. McNULTY development of two complementary humeral components lends strong support to such a model. Furthermore, partial or split humeri were reported by Murillo Ferrol (1965) and can be seen in the photographs of Summerbell (199). Inman & Saunders (1944) demonstrated patterns of referred pain from the human humerus that are strikingly similar to the pattern of loss which we have observed in the chick humerus (Fig. B). In addition, one of the major models of limb evolution proposes that metameric basals at some point in phylogeny fused to form the primitive humerus (cf. Jarvik, 1965). These observations all strongly suggest the development of a mosaic humerus. In conclusion, we have demonstrated that the development of the limb skeletal pattern may be altered by the imposition of a barrier medial to the limb field. The results of such alterations have led us to conclude that the humerus, and to some extent distal limb components, develop by the united action of several subcomponents which together make up the definitive bone. The precise time of action and nature of the axial influence upon such a pattern will be the subject of future investigation. This research was partially funded by NICHD grant HDOO836. We are particularly grateful to Drs Thomas Shepard and Alan Fantel for their assistance and to Drs Ray L. Waterson and John W. Saunders, Jr for technical advice. We are also grateful to Barbara Brownfield for help with preparation of the manuscript. REFERENCES BALCUNS, A., GASSELING, M. T. & SAUNDERS, J. W., JR (19). Spatiotemporal distribution of a zone that controls anteroposterior polarity in the limb bud of the chick and other bird embryos. Am. Zool. 1, 323. BURDI, A. R. & FLECKER, K. (1968). Differential staining of cartilage and bone in the intact chick embryonic skeleton in vitro. Stain. Technol. 43, 448. CHEVALLIER, A. (198). Etude de la migration des cellules somitiques dans le mesoderme somatopleural de l'ebauche de Paile. Wilhelm Roux's Arch. devl. Biol. 184, 53. FALLON, J. F. & CROSBY, G. M. (19). Polarising zone activity in limb buds of amniotes. In Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede), Cambridge. HAMBURGER, V. (1953). Isolation of the brachial segments of the spinal cord of the chick embryo by means of tantalum foil blocks. /. exp. Zool. 13, HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, INMAN, V. T. & SAUNDERS, J. B. C. M. (1944). Referred pain from skeletal structures. /. Nerv. Ment. Dis. 99, JARVIK, E. (1965). On the origin of girdles and paired fins. Israeli. Zool. 14, KIENY, M. (19). Sur le role du mesoderme somitique dans la differenciation de la patte de l'embryon de poulet. C.r. hebd. seanc. Acad. Sci., Paris, Serie D, 2, KIENY, M., MAUGER, A. & SENGEL, P. (192). Early regionalization of the somite mesoderm as studied by the development of the axial skeleton of the chick embryo. Devi Biol. 28, MURILLOFERROL, N. L. (1965). tude causale de la differenciation la plus precoce de L'ebauche morphologique des membres. Analyse experimental chez les embryons d'oiseau. Acta Anat. 62, 813.

15 Evidence for a metameric pattern in the chick humerus 25 SAUNDERS, J. W., JR & GASSELING, M. T. (1968). Ectodermalmesenchymal interactions in the origin of limb symmetry. In EpithelialMesenchymal Interactions (ed. Fleischmajer). Baltimore: Williams and Wilkins. SUMMERBELL, D. (199). The zone of polarizing activity: evidence for a role in normal chick limb morphogenesis. /. Embryol. exp. Morph. 5, SWEENEY, R. M. & WATTERSON, R. L. (1969). Rib development in chick embryos analyzed by means of tantalum foil blocks. Am. J. Anat. 126, {Received 8 April 198, revised 12 August 198)

16